6031-03b

Journal of Histochemistry & Cytochemistry
Production by T-cells inIntravenous Liposomal Prednisolone Downregulates In Situ TNF-Experimental Autoimmune Encephalomyelitis
Jens Schmidt, Josbert M. Metselaar and Ralf Gold
can be found at:Journal of Histochemistry & CytochemistryAdditional services and information for Volume 51(9): 1241–1244, 2003The Journal of Histochemistry & CytochemistryB R I E F R E P O R TIntravenous Liposomal Prednisolone Downregulates In SituTNF-␣ Production by T-cells in ExperimentalAutoimmune Encephalomyelitis
Jens Schmidt,1 Josbert M. Metselaar, and Ralf Gold
Department of Neurology, University of Würzburg, Würzburg, Germany (JS,RG); and Department of Pharmaceutics, Utrecht University, Utrecht, The Netherlands (JMM)
Multiple sclerosis (MS) relapses are treated with high-dose IV glucocortico-
steroids. Here we investigated mechanisms of long-circulating polyethylene glycol-coatedliposomes encapsulating prednisolone (PL) in adoptive transfer experimental autoimmune
K E Y W O R D S
encephalomyelitis. Rats received IV 10 mg/kg PL 6, 18, or 42 hr before sacrifice at disease
maximum. In formalin-fixed, paraffin-embedded spinal cord we employed a nonfluores-
cent immunohistochemical (IHC) double labeling. We stained for tumor necrosis factor-␣
(TNF-␣) in combination with a T-cell antigen. Compared with PBS-containing liposomes, PL
at 18 hr, and more at 42 hr, significantly reduced the rate of TNF-␣ double-labeled T-cells.
This correlated with an ameliorated disease score at day 5 after PL 42 hr. Our results help
to further understand mechanisms of action of drug targeting by liposomal steroids, with
possible implications for treatment of autoimmune disorders such as MS.(J Histochem Cytochem 51:1241–1244, 2003)Multiple sclerosis (MS) is a common autoimmune
ing polyethylene glycol (PEG)-coated liposomes en-
disorder of the central nervous system, with T-cell in-
capsulating prednisolone (prednisolone liposomes, PL)
filtration and demyelination as pathological hallmarks
for drug delivery of GS. This principle of drug target-
(Noseworthy et al. 2000). The main goal of therapeu-
ing improved therapeutic efficacy in a rat model of ar-
tic strategies is prevention of ongoing tissue destruc-
thritis (Metselaar et al. in press). In experimental au-
tion with subsequent permanent functional deficits.
toimmune encephalomyelitis (EAE), a dose of IV
Relapses are treated by very high-dose IV glucocorti-
10 mg/kg PL achieved much higher and prolonged tis-
costeroids (GS) (pulse therapy). The common regimen
sue levels of the GS and was superior to a fivefold
in MS involves IV therapy with 10 mg/kg methylpred-
higher dose of free GS with regard to reduced cellular
nisolone or prednisolone for 3–5 days (reviewed in
inflammation and clinical benefit (Schmidt et al. in
Brusaferri and Candelise 2000). Clinical (Oliveri et al.
1998) and experimental (Schmidt et al. 2000) data
At lower concentrations, GS effects are mainly
demonstrated that an ultra-high dose may be superior
mediated by the classical GS receptor. Only at ultra-
to the “standard” high dose of 10 mg/kg GS. Re-
high tissue concentrations are alternative nongenomic
cently, we used a novel formulation of long-circulat-
mechanisms of action “activated,” which explains thesuperior efficacy of high and ultra-high doses in thetreatment of some autoimmune disorders (Gold et al.2001).
Correspondence to: Jens Schmidt, MD, Neuromuscular Diseases
In these experiments we investigated in situ effects
Section, National Institute of Neurological Disorders and Stroke,National Institutes of Health, Building 10, Room 4N 252, 10 Cen-
of PL treatment in EAE using an immunohistochemi-
ter Drive, MSC 1382, Bethesda, MD 20892. E-mail: [email protected]
cal (IHC) double labeling method. We show that a
single IV injection of 10 mg/kg PL reduces the percent-
Received for publication March 3, 2003; accepted May 22,
age of TNF-␣-positive T-cells in the lesion as part of
1Present address: National Institutes of Health, Bethesda, MD.
the downregulation of the inflammatory response.
Our results help to understand therapeutic effects of
IV, which showed no difference compared to saline in-
liposomal GS, and ultimately may have implications
jections in previous experiments. For tissue prepara-
for treatment of autoimmune disorders such as MS.
tion, anesthetized animals were sacrificed and per-
Female Lewis rats (Charles River; Sulzfeld, Ger-
fused through the left ventricle with HAES-steril 6%
many) were 6–8 weeks old. All culture media and sup-
(Fresenius; Bad Homburg, Germany), followed by
plements were obtained from Gibco BRL (Eggenstein,
paraformaldehyde 4% in 0.1 M phosphate buffer.
Germany). Encephalitogenic T-cells for in vivo experi-
The spinal cord was removed, postfixed, dehydrated,
ments were generated and maintained as previously
described (Schmidt et al. 2000). Briefly, primed T-cells
Five-␮m cross-sections of spinal cord were deparaf-
(3 ϫ 105/ml) were restimulated with guinea pig myelin
finized and rehydrated. After blocking of non-specific
basic protein (MBP, 20 ␮g/ml) in RPMI 1640 supple-
binding with 10% BSA in 0.05 M Tris-buffered saline
mented with 1% normal rat serum, 100 U/ml penicil-
(0.15 M sodium, TBS) for 30 min, sections were incu-
lin, 100 ␮g/ml streptomycin, and 2 mM glutamine,
bated with a polyclonal rabbit anti-TNF␣ antibody
using freshly isolated and irradiated (3000 rad) thy-
(Serotec, via Biozol; München, Germany) at a dilution
mocytes (1.5 ϫ 107/ml) as antigen-presenting cells.
of 1:100 in TBS with 1% BSA, incubated overnight at
Adoptive transfer (AT)-EAE was induced by IV injec-
4C. The specificity was proved by preadsorption of
tion of 10–12 ϫ 106 freshly activated MBP-specific
the primary antibody with rat TNF-␣. Except after
T-cells in the tail vein. Animals were inspected daily by
BSA blocking, all other steps were followed by wash-
an observer masked to the respective treatment, using
ing with TBS. The primary antibody was detected
a 6 grade score: 0, healthy; 1, weight loss, limp tip of
with a biotinylated goat anti-rabbit IgG antibody
tail; 2, limp tail, mild paresis; 3, moderate paraparesis,
(Vector; Wertheim, Germany), which was pread-
ataxia; 4, tetraparesis; 5, moribund; 6, dead (Schmidt
sorbed 1:1 with normal rat serum, diluted 1:50 in TBS
et al. 2000). Disease onset in all animals started at day
with 1% BSA and incubated for 45 min. An alkaline
2 and was maximal at day 5. Bavarian state authori-
phosphatase-bound avidin–biotin complex (Dako;
Hamburg, Germany) was applied for 30 min, followed
Liposomes were prepared by the film-extrusion
by Vector red (Vector) as chromogenic substrate for
method (Metselaar et al. in press). Briefly, a lipid solu-
7–10 min. After blocking of all excess avidin–biotin
tion was prepared in ethanol containing dipalmitoyl
binding sites with an AB blocking kit (Vector), the
phosphatidylcholine (DPPC; Lipoid GmbH, Ludwig-
next primary antibodies were applied. T-cells were de-
shafen, Germany), PEG 2000–distearyl phosphatidyl-
tected with a mouse monoclonal antibody to a pan-
ethanolamine (PEG–DSPE), and cholesterol (Sigma Chemi-
T-cell antigen (B 115-1, dilution 1:500; from HyCult
cal; Poole, UK) in a molar ratio of 1.85:0.15:1.0. A
Biotechnology via Sanbio, Beutelsbach, Germany), in-
lipid film was created by rotary evaporation. The film
cubated for 1 hr at room temperature. Endogenous
was hydrated with a solution of 100 mg/ml predniso-
peroxidase activity was blocked with 3% H2O2 and
lone phosphate (Bufa; Uitgeest, The Netherlands) in
0.2 M sodium azide in methanol. The primary anti-
sterile water. The resulting lipid dispersion was sized
body was detected with a biotinylated goat anti-
by multiple extrusions through polycarbonate filter
mouse IgG antibody preabsorbed 1:1:1 with sera from
membranes to a diameter of 90–100 nm. Mean parti-
rabbit and rat at 37C for 15 min, diluted 1:200 in TBS
cle size was determined by dynamic light scattering
with 1% BSA, and incubated for 45 min. A horserad-
with a Malvern 4700 system (Malvern; Malvern, UK).
ish-peroxidase-bound avidin–biotin complex (Dako)
Phospholipid content was determined with a phos-
was applied for 30 min, followed by 3,3Ј-diaminoben-
phate assay (Metselaar et al. in press) and pred-
zidine-tetrahydrochloride-nickel (DAB-Ni, black; Vec-
nisolone phosphate concentration by reversed-phase
tor) as chromogenic substrate for 3–4 min. For each
HPLC. Each 1 ml of liposomal preparation contained
staining we added three control sections by omitting
ف4.5 mg prednisolone phosphate and an average of
either one or both of the primary antibodies. All sec-
tions were dehydrated and mounted in Vitro-clud (R.
For therapeutic studies we used prednisolone PEG
Langenbrinck; Emmendingen, Germany). In one lum-
liposomes (PL). The treatment regimen for AT-EAE
bar (intumescentia lumbalis) spinal cord cross-section,
essentially followed the protocol used in previous
an observer blinded to the respective treatment ana-
studies (Schmidt et al. 2000; Schmidt et al. in press).
lyzed 10 fields of a 10 ϫ 10 square grid at a ϫ400 en-
All experiments were performed in groups of five ani-
largement. The localization of the 10 fields followed a
mals each and were reproduced at least once, some in-
standardized graphic pattern that was applicable to all
jection time points even three times. Under general an-
sections and that yielded equal areas of gray and white
esthesia, 10 mg/kg body weight PL was injected into a
matter. Data for TNF-␣-positive T-cells were ex-
tail vein at 6, 18, or 42 hr before sacrifice at day 5.
pressed as the ratio of double-labeled T-cells and the
Negative controls received PBS-containing liposomes
total number of T-cells in percent. Statistical analysis
Liposomal Prednisolone Reduces TNF-␣ in T-cells
Treatment of AT-EAE with a single IV injection of 10 mg/
kg PL at indicated time points before sacrifice at day 5, comparedto PBS–liposomes (same experiment as in Table 1). Quantification
IHC double labeling for detection of TNF-␣ (visualized by
of IHC double labeling for TNF-␣ and T-cell markers in one lumbar
Vector red) in combination with a T-cell marker antigen (visualized
spinal cord cross-section, analyzed as detailed in the text. Percent-
by DAB-Ni, black) in a 5-␮m formalin-fixed, paraffin-embedded
age of TNF-␣-positive T-cells is given as mean Ϯ SD. Each symbol
cross-section of spinal cord from a control group AT-EAE rat at day
represents data from one rat (nϭ5 per group); data were repro-
5. Solid arrows indicate double labeled TNF-␣-producing T-cells.
duced at least once with similar results. *pϽ0.05 for PL 18 hr vs
Open arrow indicates a TNF-␣-negative T-cell. Arrowhead shows a
controls; **pϽ0.01 for PL 42 hr vs controls.
TNF-␣-producing cell, which is not a T-cell. Bar ϭ 10 ␮m.
described a novel formulation of liposomal steroids to
of the data was performed by Student’s t-test, consid-
deliver ultra-high steroid doses by drug targeting with
ering pϽ0.05 and pϽ0.01 as significant p-values.
fewer systemic doses of the free GS in treatment of
Groups of five female Lewis rats received one IV in-
EAE (Schmidt et al. in press). In these experiments we
jection of 10 mg/kg PL at 6 hr, 18 hr, or 42 hr before
observed high steroid tissue concentrations soon after
sacrifice at the peak of the disease course of AT-EAE
injection and accumulation of the liposomes in the in-
on day 5. TNF-␣ double-labeled T-cells were detected
flamed CNS. In contrast to the short-lived actions of
in spinal cord by a nonfluorescent IHC double label-
free GS, the effects of PL were clearly prolonged, and
ing technique (Figure 1). At 18 hr after the injection of
induction of T-cell apoptosis and reduction of T-cell
10 mg/kg PL, the rate of TNF-␣-producing T-cells was
and macrophage infiltration in the CNS peaked at
clearly reduced compared to controls (Figure 2). Fur-
42 hr after treatment. At this time point, a high frac-
thermore, PL at 42 hr significantly decreased the rate
tion of PL has been degraded after phagocytic uptake
of TNF-␣-producing T-cells. PL at 6 hr had no effect,
and by extracellular proteases, leading to ultra-high
which was in accord with previous findings (Schmidt
tissue levels of the active drug prednisolone. We ob-
et al. in press). In contrast to all other groups, the
served tissue levels greater than 10 moles/liter for up
peak of the disease was significantly ameliorated after
to 18 hr after PL, which is well within the range of
PL at 42 hr (Table 1), which correlated with the re-
nongenomic steroid actions and may have been opera-
duced percentage of TNF-␣-positive T cells. Experi-
tive in the reduction of the percentage of TNF-␣-posi-
ments were reproduced at least once with similar re-sults.
Treatment of AT-EAE with a single IV injection of
The therapeutic goal in treatment of MS relapses is
10 mg/kg PL at indicated time points before sacrifice at day 5,
reduction of cellular inflammation as efficiently as
compared to PBS–liposomes (same experiment as in Figure 2)a
possible to prevent ongoing tissue destruction and
axon loss. The dosing of GS as mainstay of therapy in
MS relapses is a continuous matter of debate. With re-
gard to our previous findings in EAE (Schmidt et al.
2000), one of the major issues of steroids dosing is to
reach ultra-high tissue levels, exerting multiple path-
aClinical score is given as mean Ϯ SD (nϭ5 per group). Each individual time
ways of steroid action according to a new model of
point was reproduced at least once with similar results in five independentexperiments.
steroid mechanisms (Gold et al. 2001). Recently we
bpϽ0.05 for PL 42 hr vs all other groups.
tive T-cells. Two injections of PL proved superior to
Taken together, our experiments using a nonfluo-
two injections of a free GS at a fivefold-higher dose
rescent immunohistochemical double labeling tech-
with regard to clinical and in situ effects.
nique demonstrate that a single injection of PL down-
TNF-␣, mainly secreted by T-cells and macro-
regulates the rate of TNF-␣-producing T-cells in
phages, is one of the most critical cytokines in the pro-
spinal cord of EAE rats. Analyses of the production of
cess of demyelination in the course of MS (reviewed in
TNF-␣ in situ during the demyelination vs the remy-
Steinman et al. 2002) and EAE (overview in Kassiotis
elination phase maybe useful for a better understand-
and Kollias 2001). Its proinflammatory actions have
ing of experimental treatment strategies for MS and
long been established, but during the disease course
TNF-␣ can also exert antiinflammatory properties(Steinman et al. 2002), which may explain the failure
of neutralizing TNF-␣-antibodies in therapeutic stud-
Supported by funds from the state of Bavaria, Germany.
ies of the heterogenous disease MS (The Lenercept
The invaluable technical assistance of Gabriele Köllner
Study Group 1999), and the worsening of EAE in a
and Helga Brünner is gratefully acknowledged. We thank
TNF-␣-deficient mouse model (Liu et al. 1998). More-
Louis van Bloois for his help with preparing the liposomes.
over, TNF-␣ has been shown to have neuroprotectiveproperties by promoting oligodendrocyte progenitorsand remyelination (Arnett et al. 2001). The model of a
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Volume 51(10): 1391, 2003The Journal of Histochemistry & Cytochemistry
Intravenous Liposomal Prednisolone Downregulates In Situ TNF-␣ Production by T-cells in Experimental Au-toimmune Encephalomyelitis. Jens Schmidt, Josbert M. Metselaar, Ralf Gold (in J Histochem Cytochem 51(9):1241–1244, 2003).
Figure 2, as it appeared on page 1243, contained an error of omission in the labeling of the x-axis.

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